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In vivo Atoh1 targetome reveals how a proneural transcription factor regulates cerebellar development.

ABSTRACT: The proneural, basic helix-loop-helix transcription factor Atoh1 governs the development of numerous key neuronal subtypes, such as cerebellar granule and brainstem neurons, inner ear hair cells, and several neurons of the proprioceptive system, as well as diverse nonneuronal cell types, such as Merkel cells and intestinal secretory lineages. However, the mere handful of targets that have been identified barely begin to account for Atoh1's astonishing range of functions, which also encompasses seemingly paradoxical activities, such as promoting cell proliferation and medulloblastoma formation in the cerebellum and inducing cell cycle exit and suppressing tumorigenesis in the intestine. We used a multipronged approach to create a comprehensive, unbiased list of over 600 direct Atoh1 target genes in the postnatal cerebellum. We found that Atoh1 binds to a 10 nucleotide motif (AtEAM) to directly regulate genes involved in migration, cell adhesion, metabolism, and other previously unsuspected functions. This study expands current thinking about the transcriptional activities driving neuronal differentiation and provides a framework for further neurodevelopmental studies.

SUBMITTER: Klisch TJ

PROVIDER: S-EPMC3044384 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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In vivo Atoh1 targetome reveals how a proneural transcription factor regulates cerebellar development.

Klisch Tiemo J TJ Xi Yuanxin Y Flora Adriano A Wang Liguo L Li Wei W Zoghbi Huda Y HY

Proceedings of the National Academy of Sciences of the United States of America 20110207 8

The proneural, basic helix-loop-helix transcription factor Atoh1 governs the development of numerous key neuronal subtypes, such as cerebellar granule and brainstem neurons, inner ear hair cells, and several neurons of the proprioceptive system, as well as diverse nonneuronal cell types, such as Merkel cells and intestinal secretory lineages. However, the mere handful of targets that have been identified barely begin to account for Atoh1's astonishing range of functions, which also encompasses s ...[more]

PMID: 21300888

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Project description:Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development.

Project description:Neuroblastoma is believed to arise from sympathetic neuroblast precursors that fail to engage the neuronal differentiation programme, but instead become locked in a pro-proliferative developmental state. Achaete-scute homolog 1 (ASCL1) is a proneural master regulator of transcription which modulates both proliferation and differentiation of sympathetic neuroblast precursor cells during development, while its expression has been implicated in the maintenance of an oncogenic programme in MYCN-amplified neuroblastoma. However, the role of ASCL1 expression in neuroblastoma is not clear, especially as its levels vary considerably in different neuroblastoma cell lines. Here, we have investigated the role of ASCL1 in maintaining proliferation and controlling differentiation in both MYCN amplified and Anaplastic Lymphoma Kinase (ALK)-driven neuroblastoma cells. Using CRISPR deletion, we generated neuroblastoma cell lines lacking ASCL1 expression, and these grew more slowly than parental cells, indicating that ASCL1 contributes to rapid proliferation of MYCN amplified and non-amplified neuroblastoma cells. Genome-wide analysis after ASCL1 deletion revealed reduced expression of genes associated with neuronal differentiation, while chromatin accessibility at regulatory regions associated with differentiation genes was also attenuated by ASCL1 knock-out. In neuroblastoma, ASCL1 has been described as part of a core regulatory circuit of developmental regulators whose high expression is maintained by mutual cross-activation of a network of super enhancers and is further augmented by the activity of MYC/MYCN. Surprisingly, ASCL1 deletion had little effect on the transcription of CRC gene transcripts in these neuroblastoma cell lines, but the ability of MYC/MYCN and CRC component proteins, PHOX2B and GATA3, to bind to chromatin was compromised. Taken together, our results demonstrate several roles for endogenous ASCL1 in neuroblastoma cells: maintaining a highly proliferative phenotype, regulating DNA binding of the core regulatory circuit genes to chromatin, while also controlling accessibility and transcription of differentiation targets. Thus, we propose a model where ASCL1, a key developmental regulator of sympathetic neurogenesis, plays a pivotal role in maintaining proliferation while simultaneously priming cells for differentiation in neuroblastoma.

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In vivo Atoh1 targetome reveals how a proneural transcription factor regulates cerebellar development.

Publications

In vivo Atoh1 targetome reveals how a proneural transcription factor regulates cerebellar development.

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